US11597974B2ActiveUtilityA1

Transposition of native chromatin for personal epigenomics

97
Assignee: UNIV LELAND STANFORD JUNIORPriority: May 23, 2013Filed: Jun 6, 2022Granted: Mar 7, 2023
Est. expiryMay 23, 2033(~6.9 yrs left)· nominal 20-yr term from priority
Inventors:Paul GiresiJason D. BuenrostroHoward Y. ChangWilliam J. Greenleaf
C12Q 1/6874C12Q 2537/164G16B 20/00C12Q 1/6806C12Q 1/6869G16B 30/00Y02A90/10C12Q 2521/301G16H 50/20
97
PatentIndex Score
14
Cited by
95
References
30
Claims

Abstract

Provided herein is a method for analyzing polynucleotides such as genomic DNA. In certain embodiments, the method comprises: (a) treating chromatin isolated from a population of cells with an insertional enzyme complex to produce tagged fragments of genomic DNA; (b) sequencing a portion of the tagged fragments to produce a plurality of sequence reads; and (c) making an epigenetic map of a region of the genome of the cells by mapping information obtained from the sequence reads to the region. A kit for performing the method is also provided.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
       1. A method for analyzing chromatin, comprising:
 (a) contacting chromatin in a permeabilized cell nucleus of a mammalian cell with an insertional enzyme complex such that polynucleotides of said permeabilized cell nucleus are cleaved and tagged at regions of open chromatin to produce a plurality of tagged nucleic acid fragments, 
 wherein a tagged nucleic acid fragment comprises molecular tags comprising sequencing adaptors; 
 (b) performing one or more nucleic acid reactions on the tagged nucleic acid fragments to produce a plurality of sequence reads; and 
 (c) making an epigenetic map of a region of the permeabilized cell nucleus genome by mapping information obtained from the sequence reads to the region of the genome. 
 
     
     
       2. The method of  claim 1 , wherein the insertional enzyme complex comprises two different sequencing adaptors. 
     
     
       3. The method of  claim 1 , wherein a sequencing adaptor comprises a barcode label. 
     
     
       4. The method of  claim 2 , wherein a sequencing adaptor comprises a barcode label. 
     
     
       5. The method of  claim 1 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       6. The method of  claim 2 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       7. The method of  claim 3 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       8. The method of  claim 4 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       9. The method of  claim 1 , further comprising a step (a-1) of adding a barcode label to a sequencing adaptor after step (a), carried out under conditions that prevent the tagged nucleic acids fragments from leaving the permeabilized cell nucleus. 
     
     
       10. The method of  claim 9 , wherein the insertional enzyme complex comprises two different sequencing adaptors. 
     
     
       11. The method of  claim 9 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       12. The method of  claim 1 , wherein the method is for analysis of chromatin of a single cell and further comprises a single cell sorting step (a-2) performed before step (b), and wherein step (a-2) is carried out under conditions that prevent the tagged nucleic acids fragments from leaving the permeabilized cell nucleus. 
     
     
       13. The method of  claim 12 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       14. The method of  claim 13 , wherein the insertional enzyme complex comprises two different sequencing adaptors. 
     
     
       15. The method of  claim 14 , wherein a sequencing adaptor comprises a barcode label. 
     
     
       16. A method for analyzing chromatin, comprising:
 (a) contacting chromatin in a permeabilized cell nucleus of a mammalian cell with an insertional enzyme complex such that polynucleotides of said permeabilized cell nucleus are cleaved and tagged at regions of open chromatin to produce a plurality of tagged nucleic acid fragments, wherein said insertional enzyme complex does not comprise an antibody specific to a protein that is part of said chromatin, and wherein a tagged nucleic acid fragment comprises molecular tags comprising sequencing adaptors; 
 (b) performing one or more nucleic acid reactions on the tagged nucleic acid fragments to produce a plurality of sequence reads; and 
 (c) making an epigenetic map of a region of the permeabilized cell nucleus genome by mapping information obtained from the sequence reads to the region of the genome. 
 
     
     
       17. The method of  claim 16 , wherein the insertional enzyme complex comprises two different sequencing adaptors. 
     
     
       18. The method of  claim 16 , wherein a sequencing adaptor comprises a barcode label. 
     
     
       19. The method of  claim 17 , wherein a sequencing adaptor comprises a barcode label. 
     
     
       20. The method of  claim 16 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       21. The method of  claim 17 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       22. The method of  claim 18 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       23. The method of  claim 19 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       24. The method of  claim 16 , further comprising a step (a-1) of adding a barcode label to a sequencing adaptor after step (a), carried out under conditions that prevent the tagged nucleic acids fragments from leaving the permeabilized cell nucleus. 
     
     
       25. The method of  claim 24 , wherein the insertional enzyme complex comprises two different sequencing adaptors. 
     
     
       26. The method of  claim 24 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       27. The method of  claim 16 , wherein the method is for analysis of chromatin of a single cell and further comprises a single cell sorting step (a-2) performed before step (b), and wherein step (a-2) is carried out under conditions that prevent the tagged nucleic acids fragments from leaving the permeabilized cell nucleus. 
     
     
       28. The method of  claim 27 , wherein the insertional enzyme complex comprises a hyperactive Tn5 transposase. 
     
     
       29. The method of  claim 28 , wherein the insertional enzyme complex comprises two different sequencing adaptors. 
     
     
       30. The method of  claim 29 , wherein a sequencing adaptor comprises comprise a barcode label.

Cited by (0)

No later patents cite this yet.

References (0)

No backward citations on record.